Tumor-associated glycoprotein-72 (hereinafter, referred to simply as “TAG-72”) is a mucin protein, which is a tumor-associated antigen expressed in a broad range of human carcinomas, including colon cancer, stomach cancer, pancreatic cancer, breast cancer and ovarian cancer. A murine monoclonal antibody B72.3 is the first antibody specific to TAG-72, which was developed using a membrane fraction of human breast cancer tissue as an immunogen by Dr. Jeffrey Schlom's group in NIH National Cancer Institute in the early 1980's (Colcher et al., 1981, Proc. Natl. Acad. Sci. USA, 78(5):3199-3203). Thereafter, second-generation antibodies having higher antigen binding affinity than B72.3, such as CC49 and CC83, were developed by the same research group (Muraro et al., 1988, Cancer Res., 48(16):4588-4596).
CC49 or CC83 was found to bind to more than 80% of colon cancer and about 50% of breast cancer, but rarely binds to normal tissues. Also, in vivo imaging using 131I-labeled CC49 or CC83 in cancer patients resulted in the detection of primary cancer and metastasized cancer (Divgi et al., 1994, Nucl. Med. Biol., 21(1):9-15). However, the repeated administration of the murine monoclonal antibodies to the body caused side effects or reduced therapeutic efficacy by inducing immune responses in the body. In order to minimize these undesired immune responses, humanized antibodies have been constructed by grafting complementarity determining regions (CDR) and some amino acid residues of a framework region (FR) of murine antibodies onto human antibody (Owens et al, 1994, J. Immunol. Methods, 168(2):149-65). These humanized antibodies were reported to greatly reduce undesired immune responses in patients when repeatedly administered to patients (Brown et al., 1991, Proc. Natl. Acad. Sci. USA, 88:2663).
In one study involving humanized antibodies against the TAG-72 antigen, a humanized antibody (HuCC49) of murine monoclonal antibody CC49 was developed by grafting CDRs of the murine monoclonal antibody CC49 onto light and heavy chain FRs of human monoclonal antibodies, while retaining those murine framework residues that are required for the preservation of the antigen combining-site structure (Kashmiri et al., Hybridoma 14:461-473, 1995).
U.S. Pat. No. 5,976,531 describes a Hum4 VL, VH antibody specific to TAG-72, which consists of a light chain variable region (VL), which is encoded by DNA derived from a human kappa subgroup IV germline gene (Hum4 VL), and a heavy chain variable region (VH) capable of combining with the VL to form a three dimensional structure having the ability to specifically bind to TAG-72.
U.S. Pat. No. 6,495,137 discloses a humanized anti-TAG-72 antibody or a fragment thereof, which comprises a CDR-grafted light chain having light chain CDRs of a murine anti-TAG-72 antibody grafted onto Hum4VL, wherein the murine anti-TAG-72 antibody is selected from among CC49, CC83, CC46, CC92, CC30 and CC11.
Other various humanized antibodies against TAG-72 were developed. However, there is a need to develop humanized anti-TAG-72 antibodies that have high antigen binding capacity and a reduced risk of inducing immune responses in humans.
In this regard, the present inventors, as described in Korean Pat. No. 0318761 submitted by the present inventors, identified human genes having sequences most similar to CDR and FR sequences of a murine anti-TAG-72 antibody, prepared light chain and heavy chain genes of a humanized antibody using the identified human genes, cloning the obtained genes into an expression vector, transforming a host cell with the expression vector, and cultivating the host cell, thereby developing a humanized anti-TAG-72 antibody, AKA/HzK. Compared to the humanized antibody HuCC49, the humanized antibody AKA/HzK has CDRs and FRs in which amino acid residues are replaced by amino acids more similar to those in humans and thus has reduced immunogenicity in humans, while substantially retaining antigen binding ability almost similar to that of the HuCC49 antibody. Despite this development, there is still the need for functionally excellent antibodies having a reduced risk of inducing immune responses in humans and improved antigen binding ability and affinity.
Based on this background, the present inventors, in order to prepare an antibody having excellent binding ability and affinity to TAG-72, prepared humanized heavy chain library by random mutagenesis of the CDR3 of heavy chain variable region of the humanized antibody AKA/HzK, and performed a colony lift assay using the library-expressing cells to select mutant Fab clones having high antigen binding ability. The selected clones were assessed for their antigen binding ability by competitive ELISA. As a result, a novel antibody having enhanced antigen binding ability and antigen binding affinity to TAG-72 was constructed. Further, the present inventors constructed a humanized antibody by replacing a light chain of the novel humanized antibody with a human light chain, thereby leading to the present invention.